PROJECT SUMMARY Copper (Cu) is an essential transition metal regulated by a complex set of transporters and chaperones. Mammals rely on two related p-type ATPases, ATP7A and ATP7B, to regulate intracellular Cu levels and coordinate its intestinal absorption and biliary excretion. Depending on Cu availability, ATP7A and ATP7B are actively trafficked from trans-Golgi network (TGN) to cytosolic vesicles to mediate extracellular Cu excretion. At the organismal level, ATP7A plays a required role in intestinal Cu absorption while of ATP7B is required for biliary excretion. COMMD1, the founding member of a highly conserved family that includes 9 additional members, has been long known to play a role in mammalian Cu metabolism. R ecent work from our laboratories has uncovered that COMMD1 regulates the endosomal sorting of ATP7A. This is mediated through the formation of a complex containing CCDC22 and its homologous protein, CCDC93, termed the COMMD/CCDC22/ CCDC93 or CCC complex. The CCC complex can exist in alternative configurations depending on the recruitment of specific COMMD proteins. COMMD1-CCC binds to the WASH complex, a critical regulator of receptor trafficking within the endo-lysosomal system. WASH activates the Arp2/3 complex, leading to branched F-actin deposition on endosomes, a required step for the generation of transport vesicles containing recycling cargo proteins such as ATP7A. In contrast, we recently reported that COMMD9-CCC plays an essential role in the endosomal recycling of other proteins, such as Notch. Emerging data indicates that COMMD9 also participates in ATP7A trafficking. However, it remains unclear how the CCC complex regulates endosomal sorting and in which way do different COMMD-containing CCC complexes collaborate in this process. The overall goal of this project is to provide a deep mechanistic understanding for the role of COMMD proteins in endosomal sorting and their impact in Cu homeostasis. Our hypothesis is that the primary function of the CCC complex is to regulate WASH activity. Furthermore, we hypothesize that different COMMD proteins function to localize the CCC complex to different endosomal sub-compartments to mediate cargo-specific effects and/or to regulate WASH activity along a continuum of sorting steps. The following specific aims will be pursued: (1) To uncover the mechanism by which the CCC complex regulates WASH activity. Here we will examine how CCC regulates F-Actin deposition on endosomes. (2) To understand the contribution of the COMMD9-CCC complex in endosomal trafficking. Here we will examine mechanistically how COMMD9 adds to the sorting process. (3) To assess the role of COMMD9 in cu homeostasis at the organismal level. Here we will use tissue specific Commd9 knockout mice to study the role of this gene in Cu metabolism in vivo. Altogether, the proposed studies will uncover critical aspects of vesicular sorting that have wide ranging implications in biology and human disease, as well as examine these pathways and their impact on Cu homeostasis.